mRNA Delivery Platform Based on Bacterial Outer Membrane Vesicles for Tumor Vaccine

The rapid display and delivery method for customized tumor mRNA vaccines is limited. Herein, bacteria-derived outer membrane vesicles (OMVs) are employed as an mRNA delivery platform by surface engineering of an RNA-binding protein, L7Ae. OMV-L7Ae can rapidly adsorb boxC/D sequence-labeled mRNA antigens through L7Ae-boxC/D binding and deliver them into HEK-293T and dendritic cells. This platform provides an mRNA delivery technology distinct from lipid nanoparticles (LNPs) for personalized mRNA tumor vaccination and with a Plug-and-Display strategy suitable for rapid preparation of the personalized mRNA tumor vaccine against varied tumor antigens. Key features OMVs are employed as an mRNA delivery platform through L7Ae-boxC/D binding.


Solid LB medium Reagent
Final concentration Amount Yeast extract 5 g/L n/a Tryptone 10 g/L n/a NaCl 10 g/L n/a Agar 15 g/L n/a H2O n/a 100 mL Total n/a 100 mL Prepare the solid LB medium by dissolving 1.5 g of agar, 1 g of NaCl, 1 g of tryptone, and 0.5 g of yeast extract in 100 mL of deionized water. After sterilization for 20 min at 121 °C and 0.1 MPa, add 100 μL of 50 mg/mL chloramphenicol when the temperature reaches approximately 60 °C. Add 10 mL of sterilized LB medium to a dish (10 cm) after fully mixing. After solidification, seal the solid LB medium with sealing film. Store the solid LB medium for one month at 4 °C. Note: Add antibiotics only after the temperature drops to ~50-60 °C, because their activity is seriously affected by temperature.

Liquid LB medium Reagent
Final concentration Amount Yeast extract 5 g/L n/a Tryptone 10 g/L n/a NaCl 10 g/L n/a H2O n/a 1 L Total n/a 1 L Prepare the liquid LB medium by dissolving 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of deionized water. After sterilization for 20 min at 121 °C and 0.1 MPa, store the liquid LB medium for one month at 4 °C.  5.0 with citric acid sodium citrate buffer, set the volume to 40 mL. Seal the reaction buffer for mRNA binding with sealing film and store the reaction buffer for mRNA binding for one month at 4 °C. Note: To prevent the introduction of RNA enzymes, water must be DEPC treated.

Limitations
This study preliminarily demonstrated the feasibility and effectiveness of OMVs as tumor vaccine carriers for mRNA delivery. However, compared with the LNPs-based delivery platform, the efficiency of the OMV-based delivery platform is lower, which needs to be solved urgently in the subsequent clinical transformation process. We can make efforts in the following two aspects: 1) optimize the structure of the archaeal RNA-binding protein L7Ae and the matched binding sequence boxC/D, and 2) more boxC/D sequences can be connected in series to ensure greater binding and delivery efficiency. In addition, as the bacteria-derived nanobiomaterials, the sterile production of the OMVs requires special attention in future clinical applications. In the procedure for the OMV-based nanocarriers, the bacteria were first centrifuged at 7,000× g for 15 min and almost all the bacteria were precipitated and removed. The OMVs from the supernatant were then filtered twice in 0.45 and 0.22 μm filters to ensure that the final OMVs did not contain bacteria. However, in future clinical applications, radiation sterilization of the final nanocarriers system can be performed to further ensure that the OMV nanocarriers are sterile.

General notes and troubleshooting
Troubleshooting Problem 1 There are too many or very few bacterial colonies on the plate.

Potential solution
1. This is one of the most common reasons for failure of the protocol. In many cases, the plasmid resistance genes were not associated with antibiotic, so the plasmid could not be transformed. We need to choose the right antibiotic based on the plasmid resistance genes in the backbone plasmid. 2. During competent cell resuscitation, the resistance genes were not expressed, and the LB medium with antibiotics was used (step B4), resulting in the failure of plasmid transformation. Therefore, the LB medium must be antibiotic-free in this step. 3. In the reagent setup of solid LB medium, the appropriate concentration of antibiotics was selected. If the concentration of antibiotics is too high, the target cells will not grow; if the concentration of antibiotics is too low, the growth of other bacteria will not be inhibited, resulting in the failure of plasmid transformation. In step B5, the volume of cells can be adjusted. When the volume of cells is too high, the cells will be too dense on the plate; when the volume of cells is too low, the target cells will not grow, resulting in the failure of plasmid transformation.

Problem 2
The amount of extracted OMV-L7Ae is very low. Potential solution 1. Pick the single bacterial colony on the antibiotic plate as the culture source, not from the cryopreserved bacteria in glycerin. 2. After ultracentrifugation, the OMV-L7Ae was not treated as quickly as possible, resulting in redissolution of the OMV-L7Ae in the supernatant.

Problem 3
The template DNA preparation was not successful. Potential solution Q5 high-fidelity 2× master mix is highly susceptible to inactivation by repeated freezing and thawing. On the first use, the Q5 high-fidelity 2× master mix was partitioned. Alternatively, replace the Q5 high-fidelity 2× master mix.

Problem 4
The production of boxC/D-mRNA is low in the transcription reaction.

Potential solution
Determine the correct concentration of PCR product from step D1. In the preparation, nuclease-free water was used to reduce the introduction of RNase.

Problem 5
The transfection of HEK-293T cells was unsuccessful.